Chronic lymphocytic leukemia preceded by cold agglutinin disease: intraclonal immunoglobulin light-chain diversity inV_H4-34 expressing single leukemic B cells

Several humoral autoimmune phenomena have been reported in B-chronic lymphocytic leukemia (B-CLL), including autoimmune hemolytic anemia.1 However, it remains uncertain whether these phenomena arise from the malignant cells or from residual B cells.2,3 In both possibilities, autoimmunity should develop in association with or following malignancy. On the other hand, autoimmune dysregulation is a well-known risk factor of the development of lymphoid malignancies.4B-CLL developed in a patient with existing chronic B-cell dyscrasia that had become clinically apparent as primary cold agglutinin disease (CAD),5 which is generally regarded as premalignant or low-grade malignant lymphoproliferation.6Thus, a B-CLL origin from the cold agglutinin (CA)–expressing population was likely. Because the respective autoantigens in CAD, mostly belonging to the I/i system, are widely expressed on healthy red blood cells, high antigenic pressure on the proliferating B cells may be hypothesized. Using single-cell reverse transcription–polymerase chain reaction (RT-PCR) technology, the current study examined whether B-CLL cells express immunoglobulin (Ig) receptors that share features of CAs and whether signs of intraclonal diversification and ongoing antigenic selection can be found. Notably, a light-chain–restricted, antigen-independent intraclonal diversification was found in IgM⁺/CD5⁺ B-CLL cells expressing mutatedV_H4-34/V_κA27 gene rearrangements.

A 56-year-old man sought treatment for a 1-year history of cold-dependent acrocyanosis and acral numbness. Primary CAD5 was diagnosed by the detection of high-titer (1:4000 at 4°C) monoclonal IgM/κ CA and the exclusion of CA syndrome secondary to another disease, especially lymphoma. CA specificity was determined as anti-I by reactivity against panels of group 0⁺ erythrocytes. Results of direct antiglobulin testing using anticomplement sera were positive, and mild hemolytic anemia was observed. The patient responded to the prevention of cold exposition, repeated plasma exchange, and administration of low-dose prednisolone.

During a 7-year follow-up, the paraproteinemia level increased slowly, whereas IgG serum levels (10 to 5.5 g/L) decreased. Increasing CA titers (up to 1:512 000) but moderate autoimmune hemolytic anemia (hemoglobin count, 10.4 g/dL) accompanied worsening of the acral symptoms. Platelet count was normal. Increased and sustained absolute levels of small, mature-appearing lymphocytes (6.5 × 10⁹/L-7.2 × 10⁹/L) with coarsely clumped chromatin were observed in the peripheral blood. In the bone marrow, erythroid hyperplasia and nodular infiltrates of CD20⁺/CD23⁺/CD5⁺/IgMκ⁺lymphoid cells (accounting for approximately 40% of all nuclear cells) were detected. The diagnosis of B-CLL7 was confirmed by flow cytometry and by analysis of IgV gene rearrangements.8,9 Approval was obtained from the institutional review board of the Charité University Hospital for these studies. Informed consent was provided according to the Declaration of Helsinki.

Peripheral blood mononuclear cells (PBMCs) were prepared as described10 from heparinized venous blood samples kept at 37°C. CD19⁺ B cells were enriched by negative immunomagnetic separation (Miltenyi Biotec, Bergish-Gladbach, Germany). Incubation with antibodies, propidium iodide staining, and flow cytometric analysis were performed as reported previously.10 

For analysis of IgV gene mRNA transcripts, individual CD19⁺ B cells from the expanded CD5⁺/sIgMκ⁺ population were sorted (FACSVantage, Becton Dickinson, CA) into single wells containing modified 1× RT-PCR buffer (5 mM dithiothreitol, 400 ng oligo-dT₁₈, 0.2 mM dNTP, 1% Triton, 10 U RNAsin, 40 U avian myeloblastosis virus reverse transcriptase). First-strand cDNA was generated at 42°C for 60 minutes. V_Hand V_κ mRNA transcripts were amplified by family-specific nested PCR protocols4,11,12 using 5 μL cDNA in the first round.V_κJ_κgene rearrangements were amplified from wells with clonally related heavy-chain transcripts. Moreover, the protooncogene bcl-2 mRNA was specifically amplified13 from these wells to further characterize the clonally related cells. To delineate disease stage, before and after diagnosis of B-CLL, cDNAs from metachronously collected enriched CD19⁺ B cells and single sorted B cells were subjected to specific nested PCR protocols to identify clonalIgV_H rearrangements4,11 and bcl-2 and bcl-2/IgH fusion transcripts.13,14 Following column purification, all PCR products were directly sequenced using the BigDye Termination Sequencing Kit (Perkin-Elmer, Foster City, CA) and were analyzed with an automated sequencer (ABI 377; Perkin-Elmer). Sequence alignments were performed by DNAPLOT version 2.0.1 (University of Cologne, Germany) using the VBASE Sequence Directory15 or GenBank. Analysis of mutations was performed as described.16 

The demonstration of an expanded monotypic B-cell population is central to the diagnosis of B-CLL. In this study, the diagnosis of B-CLL was confirmed by the detection of a markedly expanded CD19⁺/CD20⁺/CD5⁺/CD27⁺/sIgM⁺/CD10⁻peripheral blood population (comprising approximately 98% of all CD19⁺ cells), including a striking κ light-chain restriction (Figure 1).7-9No sIgG, sIgD, or sIgA was detected.

By single-cell RT-PCR, these leukemic B cells were found to express mutatedIgV_H/V_κgene pairings, indicating their memory cell-type origin.17,18 Twenty-eight clonally related Cμ-transcripts were analyzed, each encompassing a mutatedV_H4-34 gene segment (mutational frequency, 7.7%) rearranged to D3-22 andJ_H3 segments, with a unique 42–base pair (bp) complementarity determining region (CDR)3. Invariantly, all of theseV_H rearrangements shared the same mutations. The ratios of replacement (R) and silent (S) mutations determined for the FRs and CDRs were 4:7 (0.6) and 4:1 (4.0), respectively. A significant scarcity of FR R mutations, as observed in the patient's leukemic heavy-chain transcripts, is regarded to reflect negative selection of R mutations in these regions to maintain the functional stability of the expressed immunoglobulin molecule.16 

Coexpressed light-chain transcripts were identified in 21 of the 28 analyzed leukemic B cells. All expressed mutatedV_κA27-J_κ2 rearrangements, with the same 27-bp CDR3 lacking any indication of TdT activity. Twelve light-chain rearrangements displayed an identical mutational pattern (mutational frequency, 4.8%). The R/S ratios determined for FRs and CDRs were 5:1 (5.0) and 3:2 (1.5), respectively. Most notably, 9 rearrangements displayed additional bp exchanges in their FRs or CDRs (Figure 2a) that were confirmed by several PCR reamplifications from the original wells. These nucleotide exchanges appeared to reflect ongoing mutations because their frequency (13/5271 bp; 2.5 × 10⁻³/bp) was significantly higher than the estimated PCR error rate12(1 × 10⁻⁴/bp; P < .001; χ²analysis), and no variations were found in 7420 bp on the heavy-chain level. The intraclonal relationship of cells expressing diverseV_κA27 transcripts is given in a genealogical scheme19 (Figure 2B). To further delineate the origin of these clonally related cells, the presence of the protooncogene bcl-2 mRNA20 was analyzed by specific nested PCR and DNA sequencing. Notably, bcl-2 transcripts were detected in invariant and variant clonally related single cells in the stage of B-CLL, further indicating their relationship to the same population. By contrast, clonally related single-sorted B cells (4 of 96; 4.2% of sorted cells) obtained before the diagnosis of B-CLL did not express bcl-2 transcripts. Moreover, enriched CD19⁺ B cells obtained at the time of B-CLL were positive for bcl-2 and bcl-2/IgH fusion transcripts, whereas those collected before the time of B-CLL were negative for both products, suggesting that this transformation step occurred between these time points.20 

To our knowledge, this is the first report of intraclonal variable light-chain diversification in IgM⁺/CD5⁺B-CLL.21,22 However, given that the common and diverse mutations were randomly scattered throughout theV_κ segments lacking a preferential accumulation of R mutations in the CDRs, the ongoing mutational process seemed to be antigen independent. In this regard, our findings are in line with those of a recent study suggesting that antigen may no longer be able to play a significant role in the clonal expansion of CD5⁺/IgM⁺ B-CLL cells.22 One explanation for the variable light-chain–restricted intraclonal heterogeneity in our study might be an ongoing antigen-independent but differential mutational process affecting only the light-chain locus—for example, because of differences in accessibility or genetic abnormalities.23In this context, preliminary data show that approximately 10% of patients with B-CLL have mutations in their rearranged variable light-chain genes only.18 Whether these findings belong to a distinct subset of patients, however, remains uncertain.

Remarkably, clinical findings, shared immunoglobulin isotypes with the paraprotein, andV_H4-34/V_κIIIexpression24-26 suggest that the patient's leukemic cells had developed from his CA-expressing population. This assumption is supported by the detection of the same monoclonal and uniqueV_H4-34/CDR3/Cμ sequence in the patient's B cells obtained before diagnosis of B-CLL lacking bcl-2/IgH fusion transcripts and in B cells obtained at the stage of B-CLL with bcl-2/IgH translocation. Notably, V_H4-34expression is crucial in the specific recognition of the anti-I/i system,24-26 whereas the _HCDR3 and light chain are thought to modify the fine specificity and affinity of binding.26 Moreover, expansions of CD5⁺/CD20⁺ B cells in CAD6,27 and, conversely, B-CLL cells producing CA2 have been reported. Although antibodies using V_H4-34 do not necessarily have anti-I/i specificity25 and althoughV_H4-34 usage in IgM⁺ B-CLL cells is frequent,17,18 selection by the I-antigen might have contributed to the distinct mutational pattern of the patient's expanded CD5⁺/sIgM_κ⁺ B-cell population. In conclusion, single-cell RT-PCR provides a powerful tool for analyzing IgV gene expression in cellular (sub)populations of heterogenous lymphoproliferative diseases, such as B-CLL, to enable further insight into the association with known autoimmune phenomena.